This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include dispensing droplets to a substrate from dispensers such as pin or capillaries.(such as described in U.S. Pat. No. 5,807,522) or such as pulse jets (such as a piezoelectric inkjet head, as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere). For in situ fabrication methods, multiple different reagent droplets are deposited from drop dispensers at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array stubstrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivativesxe2x80x9d, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere
In array fabrication, the quantities of polynucleotide available, whether by deposition of previously obtained polynucleotides or by in situ synthesis, are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired target pattern, are of the correct size, and that the DNA is uniformly coated within the feature. Failure to meet such quality requirements can have serious consequences to diagnostic, screening, gene expression analysis or other purposes for which the array is being used. However, for mass production of arrays with many features, a dispensing system is required which typically has many drop dispensers.
The present invention realizes that one or more of such drop dispensers may suffer from displacement errors. By a xe2x80x9cdisplacement errorxe2x80x9d is meant a fixed error which results in a drop not being deposited in the expected location. For example, in the case where a head with multiple pulse jet is used, one or more of the jets may dispense droplets in a trajectory which is different from a normal trajectory of other of the jets. An array formed from such a head will have irregular feature spacing making interrogation of the array and/or interpretation of the resulting data more difficult. Further, in the case of large trajectory errors features may undesirably overlap to some extent. While in these situations one could chose not to use those drop dispensers in a head with displacement errors, this reduces the number of dispensers available and hence can increase fabrication time with such a head. Furthermore, this may further require monitoring of dispensers for displacement errors which increases apparatus complexity and data processing requirements during fabrication.
It would be desirable then, to provide a means by of fabricating an array with multiple drop dispensers one or more of which may have a displacement error, while still obtaining arrays with fairly regularly spaced features.
The present invention then, provides a method of fabricating an array with multiple sets of neighboring features. For each of multiple sets of neighboring features, at least one set of drops is deposited from a corresponding same dispenser onto a substrate. The result of the method is the array in which the feature sets have been formed from drops deposited by respective different dispensers (that is, the features of each set of neighboring features have been deposited by a corresponding same dispenser, with different dispensers having formed different feature sets).
The features may be of any desired moieties, such as polymers (for example, biopolymers such as polynucleotides or peptides). The drops deposited in this case may actually contain the polymers or monomers which are used to sequentially form the polymers. In the particular case where the method uses the in situ method as described above, a set of biomonomer containing drops may be deposited by the same dispenser for each feature of each of the feature sets. Any of the features in the array or within each of multiple feature sets may be of the same or different composition. For example, at least ten of the features in each of multiple feature sets may be of different moieties (such as different biopolymers, for example different biopolymer sequences).
The different dispensers for the repetitions may in one aspect, be moved in unison with respect to the substrate during deposition of respective sets of drops. Such a configuration allows the different dispensers to deposit at least some of the drops of their respective sets on a same pass of the dispensers over the substrate. The dispensers may, for example, be pulse jets (such as piezoelectric or thermoelectric pulse jets as discussed herein).
The number of different dispensers may be at least two, at least five, at least ten, at least twenty, or at least one hundred, while each feature set may have any desired number of features, for example, at least two, at least five, at least ten, at least twenty, at least one hundred or at least one thousand. Each set of neighboring features may include at least four, ten or twenty features in a non-linear configuration (that is, not on a straight line or line of constant curvature). In one particular aspect, the dispensers are operated such that a distance between at least two neighboring sets is greater than a greatest (or alternatively, average) distance separating features within the sets by; for example, at least 5%, at least 10% or at least 25 or 50% (or the foregoing inter-set distance may be at least two times, at least three times, or at least four times, the foregoing inter-feature distance). Both of these distances are measured in a same direction (that is, the distances are measured on a same straight line or parallel straight lines).
The present invention also provides an apparatus for fabricating an array useful in the methods of the above invention. In one aspect the apparatus includes a head system with multiple drop dispensers, a transport system to move the head system with respect to a substrate, and a processor which controls the head and transport system so as to execute any of the methods of the present invention. The apparatus may optionally additionally include a loading station with receptacles to retain multiple different fluids such that the dispensers can be simultaneously brought.into contact with respective receptacles for loading the dispensers with the different fluids. Each dispenser in such an apparatus may be constructed so that it holds no more than 100 xcexcl of a fluid to be dispensed as drops (or no more than 10 xcexcl or no more than 1 xcexcl of such fluid). By the dispenser holding a certain volume is referenced the entire dispenser including any reservoir in continuous communication with the remainder of the dispenser. A
The present invention further provides a computer program product for use with an apparatus for fabricating an array having multiple drop dispensers and a processor. The computer program product comprises a computer readable storage medium having a computer program stored thereon which, when loaded into the processor, performs the steps of any method of the present invention (by appropriate control of the apparatus).